Loss-of-Function ABCC8 Mutations in Pulmonary Arterial Hypertension

Abstract

Background: In pulmonary arterial hypertension (PAH), pathological changes in pulmonary arterioles progressively raise pulmonary artery pressure and increase pulmonary vascular resistance, leading to right heart failure and high mortality rates. Recently, the first potassium channelopathy in PAH, because of mutations in KCNK3, was identified as a genetic cause and pharmacological target.

Methods: Exome sequencing was performed to identify novel genes in a cohort of 99 pediatric and 134 adult-onset group I PAH patients. Novel rare variants in the gene identified were independently identified in a cohort of 680 adult-onset patients. Variants were expressed in COS cells and function assessed by patch-clamp and rubidium flux analysis.

Results: We identified a de novo novel heterozygous predicted deleterious missense variant c.G2873A (p.R958H) in ABCC8 in a child with idiopathic PAH. We then evaluated all individuals in the original and a second cohort for rare or novel variants in ABCC8 and identified 11 additional heterozygous predicted damaging ABCC8 variants. ABCC8 encodes SUR1 (sulfonylurea receptor 1)-a regulatory subunit of the ATP-sensitive potassium channel. We observed loss of ATP-sensitive potassium channel function for all ABCC8 variants evaluated and pharmacological rescue of all channel currents in vitro by the SUR1 activator, diazoxide.

Conclusions: Novel and rare missense variants in ABCC8 are associated with PAH. Identified ABCC8 mutations decreased ATP-sensitive potassium channel function, which was pharmacologically recovered.

Keywords: electrophysiology; genetics; humans; hypertension, pulmonary; ion channels.

Figure 1.

Topologic analysis of the SUR1 protein encoded by ABCC8, and sequence alignment of ABCC8 across species. Panel A shows the topology of the SUR1 protein. The 17 transmembrane segments are grouped into transmembrane domains (TMD): TMD0, TMD1, and TMD2. The two nucleotide-binding fold domains (NBF1 & 2) are indicated. Variants N72D, G111R, L135V, and E186D are located in TMD0; T229I and A240T are located in the cytoplasmic loop, L0; E791Q, D813N, and R958H are located in NBF1; R1314H and D1472N are located in NBF2. The position of each mutation is indicated by a red circle. Panel B shows the alignment of human ABCC8-encoding SUR1 protein with 14 different species, demonstrating conservation across species of each amino acid found mutated in this study.

Figure 2.

Electrophysiological consequence of SUR1 mutations on K_ATP (SUR1+Kir6.2) channel function. Whole-cell voltage clamp was used to measure expressed wildtype (WT) versus mutant K_ATP channel currents containing SUR1+Kir6.2 in COS7 cells. Panel A shows a wildtype SUR1-dependent K_ATP current trace. A voltage ramp from −120mV to +60mV over 1 second was applied every 3 seconds, from a −80mV holding potential. For all sample current traces, the vertical scale is 100 pA/pF, and the horizontal scale is 20mV. Panel B shows SUR1-dependent current traces of mutant K_ATP channels containing A240T, D813N, or D1472N SUR1 as indicated. Panel C summarizes SUR1-dependent K_ATP current densities (pA/pF) for the eight SUR1 mutants evaluated and wildtype, measured at −40mV (indicated by the black arrow in Panel A); 8 to 30 cells were studied per condition. Data are shown as means; T bars indicate standard errors. Asterisks indicate P<0.05 for the comparison between wildtype SUR1 and each mutant, as calculated by a one-way ANOVA and post-hoc Tukey test.

Figure 3.

Functional impact of SUR1 mutations on macroscopic K_ATP (SUR1+Kir6.2) channel activity. ⁸⁶Rb⁺ efflux was measured over time from COSm6 cells expressing K_ATP channels containing SUR1+Kir6.2. Panel A shows basal efflux for wildtype (WT, black curve) versus mutant (colored curves) SUR1-containing K_ATP channels, and GFP-alone (gray curve). Panel B displays the mean rate constants for K_ATP-dependent ⁸⁶Rb⁺ efflux under basal conditions. Panel C shows efflux from cells exposed to solution containing oligomycin and 2-deoxy-D-glucose to induce metabolic inhibition of cells, thereby relieving K_ATP channels from intracellular inhibition by ATP. Wildtype versus mutant SUR1-containing K_ATP channels, and GFP-alone, are compared. The inset shows exponential fits to early time points which were used to derive the efflux rate constants (see Supplementary Table S3). Panel D shows the mean rate constants for K_ATP-dependent ⁸⁶Rb⁺ efflux under metabolic inhibition conditions. For each condition, 7 to 10 cell populations were studied. Data are shown as means; T bars indicate standard errors. Asterisks indicate P<0.05 for the comparison between wildtype SUR1 and each mutant, as calculated by a one-way ANOVA and post-hoc Tukey test.

Figure 4.

Pharmacological recovery of mutant K_ATP (SUR1+Kir6.2) channels. Diazoxide restores function of K_ATP channels (SUR1/Kir6.2) containing mutant SUR1. Panel A shows rubidium efflux in the presence of diazoxide 100μM for wildtype (WT) SUR1 (black curve), mutant SUR1-containing K_ATP channels (colored curves), and GFP-alone (gray curve). Panel B shows average efflux rates for wildtype and mutant K_ATP channels in basal (gray) versus diazoxide 100μM (black) conditions. For each condition, 7 to 9 cell populations were studied. Panel C depicts whole-cell drug time courses of wildtype and selected mutant K_ATP channel currents with varying degrees of pharmacological activation. Time course depicts before drug application (gray, control), during diazoxide 100μM application (black), and during co-application of glibenclamide 10μM with diazoxide 100μM (red). The vertical scale is 25 pA/pF, and the horizontal scale is 1 minute. Arrows indicate the maximal steady-state current-density (pA/pF) achieved during diazoxide 100μM application. Panel D summarizes current density (pA/pF at −40mV) for wildtype and each mutant SUR1-containing K_ATP channel, in control (gray) and diazoxide 100μM (black) conditions; 6 to 30 cells were studied per condition. Data are shown as means; T bars indicate standard errors. Asterisks denote P<0.05 for the comparison of basal and diazoxide (Panel B), or control and diazoxide (Panel D), calculated by the paired Student’s t-test.